The past approaches described in this section could be pursued, but are not necessarily approaches that have been previously conceived or pursued. Therefore, unless otherwise indicated herein, the approaches described in this section are not to be considered prior art to the claims in this application merely due to the presence of these approaches in this background section.
Solid phase and microarray analysis involve the attachment of capture molecules, herein referred to as “entities” such as antibodies or enzymes to a support/solid matrix in a way that preserves the activity of the entity and its ability to bind to a specific target molecule. In an array, entities specific for particular targets are attached to specific identifiable locations on the matrix. After exposure to a sample suspected of containing one or more target analytes, the matrix is analyzed to determine if substances in the sample bind to the capture molecules at one or more locations on the array.
Two-dimensional microarrays have proven useful for a wide range of applications, such as in protein research. However, proteins are more difficult to attach to a solid matrix and far more complex than oligonucleotides. Thus, techniques for immobilizing a protein (antibody, enzyme, and receptor) on a microarray often require modifications compared to the more simple nucleic acid microarrays. (See, e.g., Constans, A., “The Chipping News”, The Scientist 2002.)
Many clinical diagnostic devices have been built around solid phase and microarray platforms incorporating an appropriate solid matrix to which a plurality of entities are permanently affixed to the matrix. Typically entities are attached to the surface of the solid matrix using covalent, electrostatic or hydrophobic bonding so that the entities/capture molecules remain attached to the surface during sample analysis. Target molecules that bind to the entity may be detected in a variety of ways, most commonly by attaching fluorophore tags to the target molecules. Scanners, CCD cameras or similar detectors may be used to determine the location and signal intensity of fluorescent tags bound to matrix arrays.
The amount of entity that can be affixed on a surface depends on the surface chemistry and on the nature and size of the capture molecule/entity. If insufficient amounts of entity are affixed to the surface, the resulting signal will be too weak to detect even if the entity captures or binds a tagged target molecule. Further, binding to the surface must also preserve the functional activity or property of interest of the entity.
Methods used for immobilizing entities on a support include direct covalent or electrostatic linkage of the entity to polystyrene, glass or other material; biotinylating the entity and binding it to streptavidin bound to the surface; and other similar methods. Such methods can lead to undesirable alterations of the activity or properties of the entity, which could lead to problems ranging from reduced sensitivity of an assay to inaccurate results.
Antibody based assays like Elisas and radioimmunoassay (RIAs) use antibodies or antibody fragments as attachment molecules to bind to a target. However, antibodies are costly and time-consuming to produce and screen. Even antibody fragments produced by phage display technology require a number of time consuming, iterative operations to produce a sufficient number of antibodies of a sufficient variety to test the ability of any one of them to capture an entity in a desired orientation. In addition, antibodies are large proteins, with only one or two antigen binding sites per antibody monomer. As a result, such molecules have a very high molecular weight per binding site, which potentially reduces the sensitivity of the assay.
Therefore, there is a need for a surface with a high number of binding sites per unit mass to bind and orient entities in their native form or in a desired conformation or orientation on a support material with a simple, rapid, and uniform method of producing the surface.